Vertebrate behaviors emerge from interactions of neurons across the brain, but the tools for revealing neuronal structure and function at the cellular level in living animals access only small portions of the brain. We must move toward access to structure and function anywhere in the brain of individual adult, behaving animals. In vivo three photon (3P) microscopy, a recent, but proven, technology allows optical access to deeper structures than ever before in intact mammalian brains, but much optimization remains to catalyze its wider adoption. The plan of this project is to extend the reach of 3P microscopy both within brains and through the scientific community by developing new technology and proving its worth for imaging structure and function anywhere in the brain of adult zebrafish ? a powerful vertebrate model. This project will extend the depth, speed and regional extent of imaging with 2P and 3P through a combination of technological improvements. Imaging depth will be enhanced by the development of a novel dual adaptive optics approach to correct optical aberrations that combines conjugate and standard adaptive optics to allow deep imaging through the skull with near diffraction limited resolution and improved signal. To enhance the breadth and speed of imaging, a novel approach will be developed with a light-weight, small, tandem piezo-fiber scan engine and a large field of view with the ability to raster scan any 2 to 4 sub-regions in the field. By determining and then applying optimum laser repetition rates and the best order of the nonlinear excitation as a function of depth, the number of neurons will be increased that can be imaged and reduce light exposure to improve longer term, repeated imaging through life. While the innovations will be useful for many animal models, they will be tested by imaging newly generated transgenic zebrafish lines made with CRISPR technology, as well as other established lines. The lines label neurons of different transmitter phenotype with membrane targeted fluorophores for structural imaging, or genetically encoded calcium indicators (gCaMPf or s) for functional imaging. The goal on the biological front is to provide the tools to allow the unique ability to image neuronal structure and function anywhere in the brain of an intact individual vertebrate at any time during its life, from embryo into adulthood.